Cellular senescence appears now as an important mechanism that could prevent proliferation of potential tumorigenic cells. Senescence is characterized by permanent cell cycle arrest and loss of proliferative capacity, despite continued viability and metabolic activity. Senescent cells secrete a complex mixture extracellular proteins and soluble factors, known as the senescence-associated secretory phenotype (SAPS). The senescence secretome exerts diverse effects on the microenviroment and neighboring cells. Components of the senescence secretome reinforce or implement cell cycle arrest and contribute to tumor suppression by signaling and recruiting the immune system. Among these factors, IL-6 was implicated in implementing cell cycle arrest, characteristic of senescence. A limited number of studies suggested a role for PKC in senescence, with opposite effects of different isoforms. Here our aim was to demonstrate that the epithelial specific isoform, PKCeta, plays a role in promoting senescence. Previous studies from our laboratory and other showed a role for PKCeta in the protection from cell death. We suggest that PKCeta expression in epithelial cells could shift the balance between apoptosis and senescence towards senescence thus protecting against cell death. In the present study we have also utilized a polymorphic variant of PKCeta. This polymorphic variation, the nonsynonymous SNP, changing 374V to 374I (V374I), was previously identified as a risk factor for cerebral infarction and rheumatoid arthritis in two independent genetic screens of the Japanese population. As the SNP V374I is located in the functional domain of the kinase- the ATP binding site- we have initially characterized the effect of this polymorphic change on the kinase activity of the protein. Our results show that 374I significantly enhanced the kinase activity of the protein and its phosphorylation on external protein substrates. Overexpression of PKCeta 374V and 374I in the breast adenocarcinoma MCF-7 cells enhanced the secretion of IL-6 and TNFα by LPS, with 374I having a greater effect. Oxidative stress was previously shown to induce senescence phenotype in MCF-7 cells. Oxidative stress resulted also in increased IL-6 secretion by 374I, although the 374V (WT) showed also elevated levels compared to the control treated cells. The role of PKCeta in IL-6 secretion and β-galactosidase staining was also demonstrated using PKCeta knocked-down cells by sh-RNA. Thus, our results reveal that PKCeta itself (WT) is involved in IL-6 secretion, with 374I having a more profound effect. NFkB was shown to be a master regulator of SASP by influencing the expression of NFkB target genes. Notably, our previously published studies reported a role for PKCeta as an upstream regulator of NFkB and its function in cell cycle control. One of the hallmarks of cells undergoing senescence is the involvement of the p16INK4A-Rb and p53 pathways, with p21CIP1 induction also evident. Since our previous studies showed a role for PKCeta in cell cycle regulation at the G1/S transition its effects were also determined. Upon oxidative stress, we show increased expression of p21CIP1 and p16INK4A, under conditions in which senescence is induced. Furthermore, cell expressing 374I exhibited higher levels of p21CIP1 and p16INK4A compared to 374V (WT). Taken together, our present studies reveal a role for PKCeta in establishing the senescence phenotype. There is now considerable interest in therapies that will harness senescence to control tumor promotion. Revealing the molecular regulators of senescence will improve our ability to develop new therapeutic strategies for clearing tumor cells.

Citation Format: Udi Zurgil, Assaf Ben Ari, Etta Livneh. Premature senescence is enhanced by PKCeta and its polymorphic variant V374I. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B36.